Elderly Acute Myeloid Leukemia Patients Research Articles

Identification of hub genes and potential molecular mechanisms related to drug sensitivity in acute myeloid leukemia based on machine learning.

Background: Acute myeloid leukemia (AML) is the most common form of leukemia among adults and is characterized by uncontrolled proliferation and clonal expansion of hematopoietic cells. There has been a significant improvement in the treatment of younger patients, however, prognosis in the elderly AML patients remains poor. Methods: We used computational methods and machine learning (ML) techniques to identify and explore the differential high-risk genes (DHRGs) in AML. The DHRGs were explored through multiple in silico approaches including genomic and functional analysis, survival analysis, immune infiltration, miRNA co-expression and stemness features analyses to reveal their prognostic importance in AML. Furthermore, using different ML algorithms, prognostic models were constructed and validated using the DHRGs. At the end molecular docking studies were performed to identify potential drug candidates targeting the selected DHRGs. Results: We identified a total of 80 DHRGs by comparing the differentially expressed genes derived between AML patients and normal controls and high-risk AML genes identified by Cox regression. Genetic and epigenetic alteration analyses of the DHRGs revealed a significant association of their copy number variations and methylation status with overall survival (OS) of AML patients. Out of the 137 models constructed using different ML algorithms, the combination of Ridge and plsRcox maintained the highest mean C-index and was used to build the final model. When AML patients were classified into low- and high-risk groups based on DHRGs, the low-risk group had significantly longer OS in the AML training and validation cohorts. Furthermore, immune infiltration, miRNA coexpression, stemness feature and hallmark pathway analyses revealed significant differences in the prognosis of the low- and high-risk AML groups. Drug sensitivity and molecular docking studies revealed top 5 drugs, including carboplatin and austocystin-D that may significantly affect the DHRGs in AML. Conclusion: The findings from the current study identified a set of high-risk genes that may be used as prognostic and therapeutic markers for AML patients. In addition, significant use of the ML algorithms in constructing and validating the prognostic models in AML was demonstrated. Although our study used extensive bioinformatics and machine learning methods to identify the hub genes in AML, their experimental validations using knock-out/-in methods would strengthen our findings.

Short-term Effect of Venetoclax Combined with Azacitidine and "7+3" Regimen in the Treatment of Newly Diagnosed Elder Patients with Acute Myeloid Leukemia

To compare the short-term effect and adverse reaction of venetoclax (VEN) combined with azacitidine (AZA) versus "7+3" regimen in newly diagnosed elder patients with acute myeloid leukemia (AML). From January 2021 to January 2022, the clinical data of seventy-nine newly diagnosed elder patients with AML at the Second Hospital of Shanxi Medical University and the Shanxi Bethune Hospital were retrospectively analyzed, including VEN+AZA group (41 cases) and "7+3" group (38 cases). The propensity score matching(PSM) method was used to balance confounding factors， then response， overall survival(OS), progressionfree survival(PFS) and adverse reactions between the two groups were compared. The ORR of VEN+AZA group and "7+3" group was 68% and 84%, respectively, and the CRc was 64% and 72%, respectively, the differents were not statistically significant (P >0.05). In the VEN+AZA group, there were 5 non-remission (NR) patients, 4 with chromosome 7 abnormality (7q-/-7), and 1 with ETV6 gene mutation. Median followed-up time between the two groups was 8 months and 12 months, respectively, and the 6-months OS was 84% vs 92% (P =0.389), while 6-months PFS was 84% vs 92% (P =0.258). The main hematological adverse reactions in two groups were stage Ⅲ-Ⅳ myelosuppression, and the incidence rate was not statistically different(P >0.05). The median time of neutrophil recovery in two groups was 27(11-70) d, 25(14-61) d （P =0.161）, and platelet recovery was 27(11-75) d, 25(16-50) d （P =0.270）, respectively. The infection rate of VEN+AZA group was lower than that of "7+3" group (56% vs 88%, P =0.012）. The rate of lung infections of two groups was 36% and 64%, respectively, the difference was statistically significant (P =0.048). The short-term effect of VEN+AZA group and "7+3" regimens in eldrly AML patients are similar， but the VEN+AZA regimen had a lower incidence of infection. The presence of chromosome 7 abnormality（7q-/-7） may be a poor prognostic factor for elderly AML patients treated with VEN+AZA.

Comparison of low dose cytosine arabinoside, azacitidine and azacitidine venetoclax combination treatment as remission induction in elderly acute myeloid leukemia patients

Aims: Low-intensity therapies are widely preferred in the treatment of advanced age, fragile acute myeloid leukemia (AML) patients. In this study, we aimed to compare hematological recovery rates after first cycle chemotherapy and overall survival for advanced aged AML patients treated with azacitidine (AZA) or low dose cytosine arabinoside (LDCA) or venetoclax (Ven) with AZA combination.
 Methods: Ninety-one patients were retrospectively analyzed. 
 Results: Forty-one patients treated with LDCA, 30 patients treated with AZA and 20 patients treated with AZA+Ven were included in the study. Patients who received these three treatments and who achieved response and did not receive any other treatment during the follow-up period were included in the study. Median age at diagnosis was 70. The percentage of patients who achieved neutrophil recovery after the first cycle was 27%, 73% and 50% of the patients treated with LDCA, AZA and AZA+Ven respectively. The rate of patients who achieved platelet recovery was 60%, 80%, 70% respectively. Erythrocyte transfusion independency was 54% for LDCA patients, 73% for AZA patients and 60% for combination therapy. Overall survival was longer in patients receiving AZA+Ven than other treatment groups while grade 3-4 infections were more common in the first cycle of the treatment.
 Conclusion: According to our study, patients treated with AZA had better platelet and neutrophil recovery rates with also longer overall survival than patients treated with LDCA, but total overall survival was superior in AZA+Ven combination. Hypomethylating agents with venetoclax is a preferable treatment option in elderly AML patients.

Outcome of Intensively Treated Elderly AML Patients Reported to the Harmony Alliance Compares Well to Outcome of Control Patients of the Prospective Randomized HOVON 103 Study in Elderly AML

Introduction: The evaluation of novel drugs in hemato-oncology is hampered by relatively large sample sizes needed for sufficiently powered, randomized controlled trials (RCT). External control data are increasingly applied in prospective phase II and III studies, and might be derived from Real-World Data (RWD) sources, such as national cancer registries. The HARMONY Alliance recently reported on a big data platform, consisting of data that were generated by cooperative European acute myeloid leukemia (AML) study groups ( Lang et al., Trials, 2020). HARMONY or cancer registry data might supplement prospectively collected trial control data. Here, we investigated whether and to what extent data included in the HARMONY AML database and RWD from the Netherlands Cancer Registry (NCR) compare to the control arm of the recently reported prospective randomized HOVON-103 (H103) study in elderly AML ( Ossenkoppele et al., Leukemia, 2020; Janssen et al., Leukemia, 2022). Methods: The H103 trial consecutively randomized newly diagnosed AML patients aged >65 years between standard induction chemotherapy with or without lenalidomide, tosedostat, or selinexor. External control patients concerned newly diagnosed (HARMONY: 2010-2018; NCR: 2014-2018) and intensively treated (intensified cytarabine/anthracycline) elderly AML patients aged >60 years. H103 were matched 1:1 using nearest neighbor propensity scores with NCR and HARMONY patients for age, European Leukemia Net (ELN) 2022 risk classification, and leukocyte count at diagnosis, while HARMONY patients were additionally matched for WHO performance score. Patients with missing values for propensity score factors were excluded from that analysis. The primary endpoint was overall survival (OS), calculated by the Kaplan-Meier method. Results: 67% of HARMONY patients (n=510) concerned trial patients. Median age was lower for HARMONY patients compared with H103-controls (n=279) (67 vs. 69 years, P<0.01), with also a preponderance of male patients in the H103 cohort (54% vs. 62%, P=0.03). Age and male sex did not differ between H103-controls and NCR patients (n=320) (69 vs. 69 years, P=0.46; 62% vs. 62%, P=1.0) (Table 1). WHO performance score of 0 or 1 was reported in 49% and 41% of H103-controls which was less frequent in HARMONY and NCR data (8% and 31%, P<0.01; 26% and 18%, P<0.01, respectively). Unknown WHO status was more prevalent in the external cohorts than in H103, at 43%, 54%, and 1% for the HARMONY, NCR, and H103 cohorts, respectively. ELN 2022 risk (favorable vs. non-favorable) was reported in 24% vs. 76% of H103 patients, which was similar in HARMONY patients (26% vs. 74%; P=0.73). ELN 2022 risk differed, however, for NCR patients (17% vs 83%; P=0.03). The median leukocyte count at diagnosis was lower for H103-controls than for HARMONY and NCR patients (3.65 vs. 17.1, P<0.01; 3.65 vs. 13.1, P<0.01, respectively). Propensity scores matched 278 HARMONY patients and 278 NCR patients to H103-controls. The 2-year OS (estimate±SE) was 40±3% for H103-controls vs. 35±3% for HARMONY patients (P=0.22), and vs. 30±3% for NCR patients (P=0.052), respectively (Figure 1). Conclusion: Characteristics of H103-controls and HARMONY patients appeared largely comparable, while NCR patients were more frequently ELN 2022 non-favorable risk. Following matching, OS was similar between H103-controls and HARMONY patients, while NCR patients showed inferior OS. The inclusion of a large proportion of trial patients in the HARMONY cohort, adhering to strict inclusion and exclusion criteria and fewer comorbidities compared to RWD patients, might explain these observations. These results suggest that matched data of the HARMONY Alliance might supplement prospectively collected control data in studies evaluating intensive therapy in elderly AML.

Venetoclax Plus ‘2 + 5‘ Modified Intensive Chemotherapy with Daunorubicin and Cytarabine in Fit Elderly Patients with Untreated De Novo Acute Myeloid Leukaemia: A Single-Centre Retrospective Analysis

Background: The treatment options for elderly patients with acute myeloid leukemia (AML) have been limited considering the poor tolerability of intensive standard chemotherapy. Recently, we report promising clinical outcomes in 13 untreated fit elderly patients with AML who received modified intensive induction of venetoclax in combination with ‘2 + 5‘ (daunorubicin and cytarabine) chemotherapy (modified DAV regimen), followed by venetoclax plus modified intermediate-dose cytarabine consolidation. Building on this foundation, we expanded the sample size and included 11 more patients who treated with modified DAV regimen and extended follow-up time to further explore the efficacy and safety of the modified DAV regimen for induction therapy in elderly AML patients. Method: 24 elderly AML patients (aged ≥55 years) who were received induction chemotherapy that included daunorubicin (60mg/m2 days 1-2, intravenously), cytarabine (100 mg/m 2 days 1-5, intravenously), and venetoclax (100 mg day 3, 200 mg day 4, 400 mg days 5-10, orally) from March 2021 to May 2023 were retrospectively analyzed. After induction, patients received three cycles of venetoclax (400 mg days 1-14, orally) in combination with intermediate- dose cytarabine (1 g/m 2 days 1-3, intravenously) as consolidation treatment. Overall response rate, composite complete response (CRc) rate, overall survival (OS), duration of remission (DOR), and adverse events were analyzed. Results: 24 de nove elderly patients with AML were were divided into favourable- (6), intermediate- (13), and adverse-risk groups (5) according to European LeukemiaNet (ELN) risk stratification. The cohort was consisted of 9 males (37.5%) and 15 females (62.5%) and the median (range) age of which was 64 (57-71) years. The last follow-up date was 15 July 2023, with 313 days of median follow-up. After one cycle of induction, 20 patients (83.3%) achieved CR, 2 patients (8.33%) who achieved PR after one cycle of induction attained CR after receiving a second modified DAV induction. Thus, the CR rate was 91.7% after 2-cycle induction. 2 patients (8.33%) failed to achieve remission after induction therapy. In responders, 20 of the patients were measurable residual disease (MRD) negative after 2-cycle induction, detected by multi-parameter flow cytometry (MFC). Among 22 CR patients, 20 have completed the first consolidation cycle (one patient received consolidation regimen without venetoclax due to economic pressures, and another one did not receive consoliation treatment yet), 18 patients have completed the second consolidation cycle, and 16 patients have completed all cycles of consolidation chemotherapy. Of these, 3 patients relapsed during consolidation therapy and 4 patients relapsed after completion of consolidation therapy. For the total of 24 patients, the median follow-up was 313 days. The median OS was not achieved and the median EFS was 384 days. Among the 16 patients who completed three courses of consolidation therapy, the median follow-up time was 314 days, and there were no deaths to date. The median OS was not achieved and the median EFS was 398 days. The adverse events observed during the induction therapy in most patients were Grade 3-4 neutropenia (100%), anemia (100%), and thrombocytopenia (100%). For responders in the first induction cycle, the median (interquartile range [IQR]) time to blood cell count recovery (absolute neutrophil count ≥1 ×10 9/L and platelet count ≥50×10 9/L) in patients who had a response following induction therapy was 20.0 (18.75-22.0) days. The most common non-haematological grade 3-4 adverse events observed during induction included febrile neutropenia (65.2%), pneumonia (34.8%), and constipation (15.4%). Conclusions: Our study demonstrated promising response and safety of modified DAV therapy in elderly fit de novo AML patients.

A CD38/CD28xCD3 Trispecific T-Cell Engager (TCE) As a Potentially Active Agent for the Treatment of Older Patients with Acute Myeloid Leukemia (AML)

Background: Refractory disease is common and the outcome of elderly AML patients unable to receive intensive chemotherapy with acceptable side effects remains dismal. Thus, the identification of effective and safer drugs in AML is warranted. Immunotherapeutic strategies have shown extraordinary efficacy for the treatment of hematological malignancies, such as anti-CD38 monoclonal antibodies (mAbs) in multiple myeloma. Indeed, CD38 could represent a potential therapeutic target for AML too, not only because it is expressed in AML blasts, but also because AML patients with heterogeneous or low CD38 expression could benefit from T-cell based immunotherapies that are less dependent of the density of the targeted antigen. Aims: To evaluate CD38 as a potential therapeutic target in AML, and to determine the mode of action and preclinical efficacy of isatuximab (an IgG1 anti-CD38 mAb) and SAR442257, which is a new CD38/CD28xCD3 trispecific TCE. Results: We evaluated CD38 expression in bone marrow blasts from 241 newly diagnosed elderly AML patients by flow cytometry. Overall, 99.2% of AML patients expressed CD38, 55.6% with homogeneous pattern and 44.6% with heterogeneous pattern. CD38 expression had no impact in the overall survival of AML patients. Interestingly, CD38 expression increased 2-fold in MRD positive leukemic blasts compared with patient-matched blasts at diagnosis (n=10). At 24h and up to 96h, isatuximab (70 nM) decreased CD38 surface level in 3 AML cell lines expressing low (KG-1), intermediate (MOLM-13) and high or similar (OCI-AML3) basal CD38 density as compared to AML patients. Isatuximab (70 nM) did not induce complement-dependent cytotoxicity; by contrast, we observed a trend to an increased antibody-dependent cellular phagocytosis and significant antibody-dependent cellular cytotoxicity (ADCC) only in OCI-AML3 cells in the presence of purified NK cells. In 17 primary samples from AML patients with homogeneous vs heterogeneous CD38 expression, isatuximab (70 nM) showed a median of 19% and 4.5% tumor-cell death, respectively. Altogether, these findings indicated an association between CD38 density and the mode of action triggered by isatuximab and its efficacy. As such, AML patients with heterogeneous CD38 expression are less likely to respond to isatuximab, but might benefit from TCE that are less dependent of the density of the targeted antigen. Contrary to isatuximab, the CD38/CD28xCD3 TCE did not induce a dose- and time-dependent decrease in CD38 surface levels in any AML cell line measured with a non-competing anti-CD38 antibody, regardless of the CD38 antigen density. When we cultured PBMCs from healthy adults (n=6) with MOLM-13 cells (E:T ratio of 10:1) in the presence of isatuximab or the CD38/CD28xCD3 TCE (700 pM), we observed a significantly higher tumor cell lysis at 48h induced by the latter. The same effect of the CD38/CD28xCD3 TCE was observed with purified T-, but not NK-cells from healthy adults (n=3). To identify the immune cells involved in tumor cell lysis, we confirmed that isatuximab only induced NK-cell activation, whereas the CD38/CD28xCD3 TCE induced both T- and NK-cell activation, measured as percentage of CD69+ cells. Co-culture of purified NK- or T-cells with MOLM-13 cells in the presence of the CD38/CD28xCD3 TCE demonstrated a T-cell dependent bystander activation of NK cells. Furthermore, isatuximab downregulated CD38 in a dose-dependent manner, especially in NK cells, while the CD38/CD28xCD3 TCE did not. Surprisingly, the CD38/CD28xCD3 TCE induced CD38 upregulation on T cells, indicating again T-cell activation. The efficacy of isatuximab and the CD38/CD28xCD3 TCE was analyzed in primary AML samples (n=8) and blast lysis was observed in two and three samples, respectively. Notably, two of the three CD38/CD28xCD3 TCE-responders were resistant to isatuximab. CD38/CD28xCD3 TCE-mediated lysis was independent of CD38 expression and induced no significant off-target toxicity. Conclusions: CD38 is widely present in blasts from older AML patients but nearly half show heterogeneous expression. While isatuximab-driven ADCC in AML cell lines and primary samples is dependent on CD38 density in tumor cells, the CD38/CD28xCD3 TCE exerted its anti-tumor efficacy regardless of CD38 density. Thus, AML patients expressing both high and low/heterogenous levels of CD38 could benefit from T cell based immunotherapeutic strategies targeting CD38.

Mutational Burden in High Risk Genes Do Not Affect Remission Rates and MRD Clearance in Elderly AML Patients Receiving CPX-351 Induction

Background: CPX-351 has recently been approved for the treatment of patients diagnosed with Acute Myeloid Leukemia (AML) arising from a previous myelodisplastic syndrome (s-AML) or secondary to chemotherapy (t-AML) as per former WHO 2016 classification. Minimal residual disease (MRD) assessment is a strong prognostic factor for relapse and shorter survival in AML patients undergoing intensive induction chemotherapy. Recent studies explored the role of genomic profile of AML influencing clinical outcome. In particular, Papaemmanuil et al showed that mutations in critical genes such as TP53, ASXL1, SRSF2 and RUNX1 are independently associated with a worse prognosis; moreover, co-occurrence of different high-risk mutations are predictors of dismal outcome when conventional chemotherapy is administered. Adverse risk mutations are particularly frequent in s-AML and t-AML. As clinical trials evaluating the prognostic relevance of MRD in the context of different molecular subsets mainly involve younger patients affected by de novo AML and receiving conventional 3+7 chemotherapy, there is a lack of data to define the impact of specific mutations on MRD clearance and prognosis in elderly s- AML and t-AML patients receiving CPX-351 induction. Aims: The aim of this study was to evaluate the prognostic impact of recurrent genic mutations and mutational burden at diagnosis on CR probability, MRD clearance and prognosis in a cohort of elderly s-AML or t-AML patients treated with CPX-351 induction chemotherapy. Methods: The study included 67 elderly patients (>60 year, median age 69, range 60-77) with a diagnosis of s-AML or t-AML according to WHO2016 classification, treated in our Center with CPX-351. Next generation sequencing (NGS) was performed using the Myeloid Solution panel by SOPHiA Genetics, encompassing 34 critical genes mutations. Sample have been processed on a Illumina MiSeq platform and the analysis performed with SOPHiA DDM® Software. MRD was analysed in all patients achieving complete remission (CR) with multicolour flow-cytometry, with a threshold of 0.1%. Results: AML patients were re-classified according to ELN 2022 risk score which was adverse, intermediate and favourable in 35, 29 and 3 patients, respectively. Median number of mutations for single patient (mutational burden) by NGS was 4 (range 2-9). Most frequent mutations involved: RUNX1 (44%), ASXL1 (37%), TP53 (33%), IDH1 (18%), IDH2 (14%), DNMT3A (14%), TET2 (8%). Four patients died before response assessment, mainly due to infections. Fifty-four patients (81%) achieved complete remission (CR) after first induction cycle; MRD evaluation was negative in 28/54 responding patients (52%). Univariate and multivariate analysis showed that factors traditionally associated with poor outcome such as ELN risk group, leukocytosis at diagnosis or previous treatment with hypomethylating agents did not affect the probability of CR rate and MRD negativity. Interestingly, having any high risk mutation, the combination of ≥2 high risk mutations or high mutational burden (having ≥ 4) did not affect CR probability and MRD negativity rate both in univariate and multivariate analysis. After cycle 2, the cumulative CR rate was 56/67 (84%), with 33/56 (59%) responding patients obtaining MFC MRD negativity. After a median follow up of 36 months (CI 95%; 22.4 -48.5 months), median OS was 19 months (CI 95% 13-24), whereas 2 year OS was 24%. Multivariate OS analysis revealed that negative MRD was the strongest independent predictor of longer survival (p<0.05). Notably, OS was not affected by mutational burden (29.2% and 27.6% for patients with less or more than 4 mutations, respectively, p=n.s, Fig.1). In landmark analysis, patients achieving CR and proceeding to allogeneic stem cell transplantation consolidation (HSCT) within 3 months from CR (N=13) had a significantly better outcome if compared to CR patients who did not receive HSCT or proceeded to transplant later (N= 41, 2-year OS: 84.6% and 31.7, respectively, p<0.03, Fig. 2). Conclusion : CPX-351 is able to induce good quality remission with high CR rate and MRD negativity, regardless of mutational burden, allowing a high number of elderly AML patients to undergo to HSCT. MRD evaluation has proven to be a strong prognostic tool also in the setting of elderly s-AML and t-AML patients receiving CPX-351. The prognostic value of high risk mutation seems to be less relevant in CPX-351 treated patients.

Acute Myeloid Leukemia Driven IL-3 Dependent Upregulation of BCL-2 in Non-Malignant Hematopoietic Progenitor Cells Increases Venetoclax Induced Cytopenias

The BH3 mimetic venetoclax, in combination with low dose cytarabine, decitabine or azacitidine has shown clinical efficacy in newly diagnosed acute myeloid leukemia (AML) (1, 2). This has been a significant advance, particularly in the treatment of older AML patients, and those who are ineligible for intensive chemotherapy, who historically have been difficult to treat. Venetoclax selectively inhibits the BCL-2 protein which is overexpressed in AML in order to activate intrinsic apoptosis. However, this treatment regime is associated with cytopenias including neutropenia, febrile neutropenia, and thrombocytopenia which leaves patients immunocompromised (3). The underlying cause of cytopenias in the context of venetoclax-treated AML remains unexplained. Here we describe a mechanism for venetoclax-induced cytopenias in AML. Two established AML models were used to assess BCL-2 expression profile in HPCs. Isolated LSK cells were co-cultured with mouse AML (HOXA9/Meis1 or MN1 overexpressing cells). RT-qPCR analysis identified BCL-2 upregulation in LSKs co-cultured with both AML cell types. To understand the significance of this in vivo, MN1 cells were engrafted into C57BL/6 mice and LSKs were FACS purified. Analysis confirmed increased BCL-2 gene and protein expression by RT-qPCR and flow cytometry when compared to control LSKs. Next, to recapitulate the clinical cytopenias, MN1 engrafted mice or control mice were treated with venetoclax for 7 days by oral gavage. Flow cytometry analysis of bloods and bone marrow in venetoclax-treated AML mice determined neutrophil depletion and reduced numbers of HPCs, namely LSK, MPP, HSC and GMP, compared to untreated AML mice . Todetermine the mechanism of BCL-2 upregulation in LSKs we explored various signalling pathways using targeted inhibitors on LSKs cultured with MN1 conditioned media. Using this method, we established that interleukin 3 (IL-3) activates BCL-2 transcription in LSKs leading to a dependency on pro-survival BCL-2 protein. Furthermore, treatment of LSKs, cultured in MN1 conditioned media with IL-3 neutralising antibody MP2-8F8 reduced BCL-2 gene and protein expression in LSKs. Finally, MN1 engrafted mice were treated with venetoclax and IL-3 neutralising antibody in combination which restored neutrophil levels to numbers observed in untreated AML mice. Here, we demonstrate that BCL-2 is overexpressed in non-malignant HPCs during AML progression using two AML models. In addition, using our syngeneic mouse model we show that venetoclax induces HPC depletion. In vitro studies confirm that IL-3 mediates BCL-2 upregulation in HPCs - this sensitises HPCs to venetoclax and causes cytopenias. We also demonstrate that neutrophils can be recovered using IL-3 neutralisation in combination with venetoclax in AML engrafted mice. Taken together, these findings provide biologic insight for IL-3 inhibition alongside venetoclax to reduce the incidence of cytopenias observed in elderly AML patients.

Novel Gene Mutations Associated with Disease-Free Survival in Elderly Patients with Acute Myeloid Leukemia Receiving First-Line Induction-Consolidation with or without Azacitidine Post-Remission Treatment

Background: The achievement of complete remission (CR) is an important milestone for patients with acute myeloid leukemia (AML) undergoing curative-intent therapy. However, patients who achieve CR with induction therapy for AML will relapse within months. Therefore, research is focused on prolonging leukemia-free survival (DFS). We have recently reported on the randomized phase III study to compare the efficacy and safety of azacitidine (AZA) versus best supportive care (BSC) for treatment of AML in elderly patients who achieved first CR after a homogeneous intensive induction and consolidation phase (QoLESS AZA-AMLE, Oliva et al. Cancers 2023). At 2 and 5 years, DFS was 6.0 (95% CI:0.2-11.7) months in the BSC arm vs. 10.8 (95% CI:1.9-19.6 p=0.23) months in the AZA arm. We report on the ancillary translational study (QOL-ONE Trans-2) to evaluate biological changes in bone marrow samples through Next Generation Sequencing (NGS). Objective: In patients enrolled in the trial who reached CR and were randomized to receive AZA or BSC, the endpoint of the present study was to evaluate the effect modification by gene mutations at AML diagnosis on the relationship between treatment allocation and relapse (disease-free survival, DFS). Methods: The study was performed on available biological samples collected at baseline, randomization, and 6-month post remission. DNA was subjected to high throughput NGS using genes commonly mutated in myeloid malignancies and prepared with a home-set of 350 genes for Illumina (University of Kyoto, Japan) for all subjects with available vials. Only variants with high-quality reads, were considered. A Variant Allele Frequency (VAF) ≥ 4% was considered an appropriate threshold for minimal burden of clonality to be reported. Results: Samples were available for 24 patients, 12 males, of median age 71 (65-74) years, 11 allocated in AZA arm and 13 in BSC arm. At diagnosis, mutations were detected in all patients with a range of 5 to 17 (median 10) simultaneous mutations, the most frequent being DNMT3A (42%), NPM1 (33%) and TET2 (33%). The most frequently mutated genes at diagnosis are represented in the figure. Only FANCA gene, mutated in 4 patients was associated with a hazard risk (HR) 4.96 (95% CI 1.34 - 18.35; p=0.02) of relapse and, interestingly, HLA-A, which was mutated in 7 patients, was associated with a HR 0.277 (95% CI 0.08 - 0.95; p=0.049). Patients with HLA-A mutation had a significantly longer survival (Figure 2). Three HLA-A mutated patients were allocated in 5-Aza arm and 4 in BSC arm. In a multivariate COX model, the effect of baseline HLA-A mutation on DFS is independent of the allocation arm (P=0.041). HLA Class I and Class II alleles were assessed based on the Luminex xMAP technology: for all cases, the HLA-SNPs are located on chromosome 6 in exon 2, 3 and 4 within protein-codifying sequences. Conclusions: HLA gene mutations are not usually investigated for diagnostic or prognostic purposes in AML. The extensive 350-gene panel used in this study included HLA genes and has revealed a possible favorable role of HLA-A polimorphisms for the prognosis of elderly patients with AML undergoing intensive chemotherapy. Further specific HLA NGS analyses are undergoing to determine whether this concerns genuine somatic mutations. The present finding is worthy of further investigation to confirm its value in prognosis and its therapeutic implications.

Efficacy of Primary Prophylaxis in Patients with MDS or AML Treated with Non-Intensive Agents: Systematic Review with Meta-Analysis

Background: Infections are common among patients with myelodysplastic syndromes/neoplasms (MDS) and acute myeloid leukemia (AML) due to neutropenia, which leads to increase in hospitalization, morbidity and mortality. Antibiotic prophylaxis has been proven to reduce the incidence of infections in oncologic and hematooncologic patients receiving intensive chemotherapies. Accordingly, most hematology and oncology societies advice for its use especially in high-risk patients. These recommendations, however, cannot be directly extrapolated to elderly MDS or AML patients treated with less intensive regimens such as single agent hypomethylating agents (HMA) or low-dose cytarabine (LDC), as controlled prospective randomized trials are not available in this context. Aim: The aim of this systematic review was to summarize published data on the efficacy and safety of primary antibiotic prophylaxis in patients with MDS or AML treated with non-intensive agents and perform a meta-analysis. Methods: We performed a systematic review using PubMed and Embase databases on 14th September 2022 without time-restrictions concerning publication data. Inclusion criteria comprised demographics (adult), type of study (retrospective and prospective cohort studies, case control studies, randomized controlled trials), study population (patients with MDS, MDS/MPN or AML), and treatment (hypomethylating agent (HMA), low-dose cytarabine (LDC)). Study outcomes/endpoints included information on efficacy (reduction of infection or fever episodes requiring antibiotic treatment (bacterial, viral, fungal), hospitalizations, intensive care unit admissions or leading to dearth) and safety (antibiotic side-effects and resistance) of primary antibiotic prophylaxis. Meta-analysis was performed and odds ratios with 95% confidence intervals shown in a Forrest Plot. Results: From 233 screened studies, we identified no randomized or prospective studies and only two retrospective studies with 68 patients and 346 treatment cycles that matched our inclusion/exclusion criteria. ( A). One study included AML (n=40, Bainschab et al, 2016) and the other MDS patients (n=28 , Lee et al, 2011). Patients recived primary antibiotic prophylaxes in 79/215 (25%) and 95/131 (72.5%) treatment cycles, respectively (B). The antibiotics used included fluorquinolones (92.5% and 95%), penicillins (6.5% and 0%) and cephalosporins (1% and 5%). Infections episodes were reported in 53/215 (25%) and 15/131 (11%) treatment cycles. A meta-analysis of the selected studies suggests a significant benefit in reducing the incidence of infection or febrile episodes (OR 0.43 [0.25-0.75], p<0.003) with potential reduction in hospital admission, especially in patients with low ANC (< 0.5 G/l). As additional risk factors for infectious complications we identified severe cytopenia (grade ≥3) in at least one lineage, earlier therapy cycles, transfusion dependency, increased LDH and high Charlson Comorbidty Index. Data regarding the safety and tolerability of antibiotic prophylaxis was not available. Conclusion: Our investigation with only few eligible studies, patients and treatment cycles underlines an unmet need for prospective controlled trials in this field.Our systematic review with meta-analysis suggests a significant benefit of antibiotic prophylaxis in MDS and a tendency in AML patients undergoing single agent HMA or LDC treatment. Those individuals with high-risk features seem to have the greatest benefit. Prospective, controlled and randomized trials are, however, lacking and should be encouraged, even more as combinations with venetoclax are becoming the standard of care in AML and potentially also in MDS with higher risk of neutropenic infekctions.

SIE/Sies/GITMO Criteria in Elderly Patients with Acute Myeloid Leukemia (AML): Useful Also in the New Drug Era? a Multicentric Real-Life Study By the Hematological Network Rete Ematologica Lombarda (REL)

Introduction: In elderly acute myeloid leukemia (AML) patients (pts) the debate about the use of intensive chemotherapy (iC) as opposed to non-intensive therapy (niT) or best supportive care (BSC) is a hot topic, particularly after the introduction of venetoclax (VEN) use. Age, comorbidities, functional impairment, therapy benefits and risks, pts preferences, and AML features influence this choice. The SIE/SIES/GITMO (“fitness”) criteria (Ferrara, 2013) are a valuable and comprehensive tool, but a revalidation is needed in the light of the new treatment options. Methods: Within the Rete Ematologica Lombarda (REL), we evaluated 1) the concordance between “fitness” of pts, and the type of treatment they actually received, 2) the overall survival (OS) according to the “fitness”, to the prognostic European Leukemianet (ELN17) stratification and to the treatment received, and 3) the presence of other parameters, as Charlsons Comorbidity Index (CCI), potentially useful in therapy decision. Pts were classified as fit to iC (FIT), unfit to iC (UNFIT), or unfit even to niT (FRAIL), as defined by the SIE/SIES/GITMO criteria. Results: From Jan 20 to Dec 22, 503 AML pts (53% de-novo), with a median age of 76 years (y) (range, 65-93) were diagnosed consecutively at 11 Hematology Units. According to “fitness” criteria, 25% of pts were FIT, 61% UNFIT, and 14% FRAIL (Table1). Median age was significantly lower in FIT pts (70 y), as compared to UNFIT (78 y) and FRAIL (79 y) (p <0.0001). Overall, the concordance between “fitness criteria” and the treatment actually received by pts was 75.9% (71% in FIT, 76% in UNFIT and 84% in FRAIL). After a median follow-up of 12 months (m), median OS of the whole population was 7.4 m. Median OS of FIT, UNFIT and FRAIL pts was 13.7, 7.2 and 1.4m, respectively (p <0.0001, Fig 1a). According to physicians' decision, 18% of pts received iC, 55% niT, mainly hypomethylating agents (HMA: azacytidine or decitabine) + VEN, and 27% BSC. Median OS was 17m, 10.2m, 1.4m in iC, niT and BSC pts, respectively (p<0.0001, Fig 1b). According to ELN17, evaluable in 73% of pts, 19% were favorable (fav), 20% intermediate (int) and 61% adverse (adv), without differences into “fitness” groups. Median OS was significantly worse in ELN adv than in ELN fav and int (p: 0.001 and p: 0.002, respectively, Fig 1c). In FIT pts, median OS was 17m with iC and 11.4m with niT (VEN-HMA) (p: 0.3). The use of iC was associated with younger age (median age: 69 in iC vs 73 y in niT, p<0.0001), lower CCI (CCI< 2: 88.5% in iC vs 64% in niT, p: 0.0032) and not significantly with better ELN risk (ELN adv 49% in iC pts vs 70% in niT, p: 0.07). The use of iC or niT did not affect remission rate (64% vs 71%) nor OS in different ELN risk groups. In UNFIT pts, median OS was 14m with niT and 3.8m with BSC pts (p<0.0001). The use of niT compared to BSC was associated with younger age (median 77 vs 81 y; p<0.0001), not adv ELN risk (41% vs 7%, p: 0.0001), lower ECOG performance status (PS) and CCI (PS<2: 81% vs 65%, p: 0.0001; CCI<2: 66% vs 50%, p: 0.038). In UNFIT pts treated with VEN-HMA median OS was 11m vs 7.3m with HMA only (p: 0.019). The use of VEN-HMA rather than HMA only was associated with younger age (median: 76 vs 80 y; p: 0.0045), better PS and CCI (PS<2: 94% vs 84%, p: 0.026; CCI<2: 68% vs 50%, p: 0.008). ELN risk distribution was similar. In non-adv risk pts, OS was 17m with VEN-HMA, significantly better compared to HMA only (7m) (p: 0.03) as well as to adv risk pts treated with VEN-HMA (p: 0.0017). Age >82y, number of comorbidities, lack of caregiver were the most frequent reasons for the physician's preference for HMA therapy over VEN-HMA. InFRAIL pts, median OS was 2.9 m with niT and 1.1m with BSC pts (p: 0.04). The use of niT was associated with a relatively better PS and younger age (<73 y). At multivariable analysis, ELN fav and iC favorably affected survival, whereas a FRAIL fitness status, PS>2, and CCI>2 proved to be independent adverse prognostic factors. Conclusion: Fitness stratification of SIE/SIES/GITMO correlated to pts's survival and were useful in the decision-making approach of treatment also in the new drugs era. Within the “fitness” categories, age, CCI and particularly ELN risk, had an impact on the choice of the type of induction therapy, in FIT pts (iC vs niT) without changing the outcome, while in UNFIT pts an under-treatment worsened OS. These preliminary data should be implemented with a longer follow-up and a larger sample size.

Targeting G3BP2 Sensitizes Acute Myeloid Leukemia to Venetoclax Treatment Via ELF1-MCL1 Axis

Venetoclax-based combination therapies have emerged as promising treatments for elderly acute myeloid leukemia (AML) patients. However, venetoclax resistance renders the treatment ineffective, mainly due to the upregulation of MCL1. Thus, new venetoclax-based combination therapy is under development to circumvent venetoclax resistance. The Ras-GTPase-activating protein SH3-domain binding protein 2 (G3BP2) is upgraded in several solid tumors. Targeting G3BP2 re-sensitized tumor cells to chemotherapy. To date, whether targeting G3BP2 sensitizes venetoclax treatment in AML and its role in leukemogenesis has not been defined. To this end, the primary AML cells and AML cells were collected and cultured with compound C108 (G3BP2 inhibitor) and venetoclax alone or both. The result showed that co-culture with compound C108 and venetoclax decreased the cell viability in primary AML and AML cells, indicating the potent synergy of G3BP2 inhibitor and venetoclax. The Quantitative real-time PCR (RT-qPCR) showed that G3BP2 was overexpressed in AML patients and was associated with poor prognosis. Knockdown (KD) of G3BP2 decreased proliferation and induced apoptosis in AML cells. In addition, G3BP2 inhibition impeded the tumorigenicity in the subcutaneous AML xenograft tumor model. These data demonstrated that G3BP2 sensitized AML to venetoclax treatment and is critical in AML development. The RNA-seq analysis was performed to investigate the role of G3BP2 in venetoclax resistance and leukemogenesis. The results showed that differentially expressed genes (DEG) were enriched in apoptotic processes when G3BP2 were downregulated. The immunoblotting results indicated that G3BP2 knockdown decreased the expression of MCL1 and BCL2, and vice versa. Moreover, we found that the transcription factor ELF1 was positively correlated with G3BP2 by the immunoblotting and RNA-seq analysis. The result was further validated by the Pearson correlation analysis based on the online database. The expression of ELF1 was decreased when G3BP2 were downregulated. Thus, we concluded that G3BP2 is an upstream gene of ELF1 and MCL1. To further investigate the relationship among G3BP2, ELF1, and MCL1, the RNA binding protein immunoprecipitation assay (RIP) and mRNA stability analysis was performed. The results suggested that G3BP2 interacted with the ELF1 mRNA to enhance its stability. We employed bioinformatics analysis via the JASPAR database and RPISeq tools and found ELF1 may facilitate the transcription of MCL1, which was validated in the following study. The expression of MCL1 was restored in the G3BP2 KD cells when ELF1 was overexpressed. We subsequently performed CHIP-qPCR, dual-luciferase reporter gene assays with truncated MCL1 promoter fragments. A 576bp of the binding region upstream of MCL1 promoter was defined, indicating that ELF1 regulated MCL1 expression by modulating its transcription activity. Taken together, our data suggested that targeting G3BP2 decreases MCL1 expression by disturbing ELF1 mRNA stability, thus increasing the sensitivity to venetoclax treatment. We finally established the intra-venous tumorigenesis xenograft model to verify the role of G3BP2 downregulation in venetoclax-treated leukemia mice. The NCG immunodeficient mice were transplanted with U937 cells and administrated with venetoclax daily from 7-21 days post transplantation (Figure 1A). The data showed that G3BP2 KD in combination with venetoclax reduced clinical scores and prolonged the overall survival of AML mice (combination vs. venetoclax: p<0.01; combination vs. G3BP2 KD: p<0.05, Figure 1B). The reduced leukemic burden was observed when targeting G3BP2 in combination with venetoclax. Meanwhile, we verified the decreased expression of ELF1 and MCL1 in mice targeting G3BP2 in combination with venetoclax by immunofluorescence (IF) and flow cytometry analysis. These data supported the synergic effect of G3BP2 inhibition and venetoclax treatment in AML. Our findings emphasize the critical role of G3BP2 in conferring resistance to venetoclax in AML by stabilizing ELF1 mRNA to regulate MCL1 expression. Targeting G3BP2 represents an appealing therapeutic strategy for patients with venetoclax-resistant AML.

Preclinical Evaluation of Bisantrene As Single Agent and in Combination with Decitabine for Acute Myeloid Leukemia

Anthracyclines, particularly doxorubicin and idarubicin, together with cytarabine, have comprised standard of care induction chemotherapy in acute myeloid leukemia (AML) for almost 4 decades. Although 60% of patients achieve remission, the majority relapse within 1-2 years. As such, outcomes in AML remain poor, with overall 5-year survival around 27%. In elderly AML patients the situation is worse, with 80% dying within one year of diagnosis. Toxicity of induction therapy is a major barrier to treatment success, precluding many unfit and elderly patients. Hypomethylating agents (HMA; azacytidine, decitabine) provide a less toxic alternative and have improved treatment options for the unfit. However, only 50% of patients respond to HMA therapy and most eventually acquire resistance, resulting in relapse. Approaches that reduce the toxicity of induction therapy and improve the efficacy of HMA treatment are emerging, e.g. combinations with Venetoclax. Nevertheless, novel combination strategies that improve outcomes in certain mutation subtypes need to be explored. Bisantrene, an anthracene derivative developed over 4 decades ago as a less cardiotoxic chemotherapy alternative to anthracyclines, shows broad activity across multiple solid and haematological malignancies. Several clinical studies in the 1980s showed bisantrene is an effective AML salvage therapy, producing response rates up to 50% without accompanying cardiotoxicity (Rothman J., 2017 Int J Cancer Res Ther;2(2):1-10) In a recent investigator-led Phase II trial of heavily pre-treated relapsed/refractory AML patients, 1/10 achieved a complete remission and 3/10 patients achieved partial remission. Interestingly, all responders were characterised by extramedullary disease (Cannani et al., 2020 Eur J Haematol 2021;106:260-266). We postulate that bisantrene may represent a suitable candidate for combination with HMA in patients unfit for standard induction chemotherapy. To explore this idea, we evaluated the in vitro and in vivo effects of bisantrene both alone and in combination with decitabine. The in vitro activity of bisantrene was initially assessed in a range of human and mouse AML cell lines covering the major molecular and clinical subtypes. All cells were sensitive to bisantrene, showing IC 50 values in the range 16-563 nM. FLT3-ITD + cells in particular were sensitive, while NRAS, KRAS and KIT mutant lines were also responsive. When tested against primary patient AML samples, 15/49 (30.6%) responded strongly to bisantrene treatment ex vivo over the dose range 0.1-1 µM. Bisantrene was shown to induce G1 accumulation and dose-dependent apoptosis in the FLT3-ITD + cell line, MOLM13. When tested in combination with decitabine, pre-treatment with decitabine for 24 h followed by decitabine plus bisantrene for 3 days produced synergistic cytotoxicity in MOLM13, MV4-11 and OCI-AML3 cells. Bisantrene and decitabine together resulted in S phase accumulation in MOLM13 cells and synergistic induction of apoptosis. The in vivo efficacy of bisantrene alone and in combination with decitabine were examined using the MOLM13- luc cell line and a patient derived xenograft (PDX; AML16) (FLT3-ITD +, NPM1, IDH2 and WT1 mutant) in NSG mice. Bisantrene (10 mg/kg i.v. twice/week) significantly reduced leukemic burden and increased median survival compared to vehicle control in MOLM-13- luc engrafted mice, and significantly reduced leukemic burden and increased median survival in a dose and schedule-dependent manner (2.5mg/kg and 5mg/kg, twice or three times weekly) in the PDX model. The combination of bisantrene (5 or 10 mg/kg twice weekly) and decitabine (0.2 mg/kg i.p. 5 days/week) significantly improved survival in MOLM13- luc (Figure 1) and PDX AML16 (Figure 2) engrafted mice, respectively, relative to vehicle control and each single agent. The combination also reduced leukemic infiltration to extramedullary sites (spleen, liver, uterus, brain). These data support that addition of bisantrene to HMA backbone therapy may provide an important new treatment strategy in AML. A prospective Phase Ib/II trial is being planned to evaluate this new therapeutic approach.

A Systematic Review of Venetoclax and Azacitidine: Effectiveness and Safety for Newly Diagnosed and Relapsed Acute Myeloid Leukemia Patients

Introduction: Acute Myeloid Leukemia (AML) remains the most prevalent acute leukemia in adults, often associated with a bleak prognosis. Current treatment recommendations for elderly AML patients incorporate a combination of venetoclax (Ven) and azacitidine (AZA) following trials that underscored Ven's efficacy. This review seeks to analyze and summarize the literature concerning the effectiveness and safety of the Ven + AZA regimen for adult patients with newly diagnosed (ND) and relapsed/refractory (R/R) AML, and those unsuitable for intensive chemotherapy. Materials and Methods: Following PRISMA guidelines, a comprehensive electronic literature search was conducted across PubMed, Embase, Clinicaltrials.gov, Cochrane Library, and Web of Science from inception to 2023. MeSH terms and relevant keywords for (Leukemia, Myeloid, Acute) AND (Venetoclax) AND (Azacitidine) were used. From 327 initially identified articles, 12 studies were selected for inclusion, and citation searching further added 2 studies, summing up to 14. We analyzed data from a combined patient pool of 1256. Results: The review encompassed 13 non-randomized and 1 randomized clinical trials. The median patient age was 75.2 years. Primary outcomes comprised complete remission (CR) and complete remission with incomplete count recovery (CRi), yielding a pooled CR/CRi of 57.8%. Secondary outcomes included an overall response rate (ORR) of 61.1%, and a median overall survival (mOS) of 8.94 months with a median follow-up duration of 13.25 months (Table 1). Notably, high-grade adverse effects were predominantly hematological (anemia, neutropenia, thrombocytopenia, febrile neutropenia) while low-grade effects primarily involved gastrointestinal symptoms (nausea, vomiting, diarrhea, constipation, decreased appetite, etc). Conclusion: The current data suggest that the Ven + AZA regimen is both efficacious and well-tolerated for treating newly diagnosed and relapsed/refractory AML. Cytopenias across all three cell lines emerged as the most reported adverse effects of Ven + AZA therapy. Ongoing Phase III clinical trials are expected to enhance our understanding of this regimen's efficacy and safety profile, solidifying its role in standard AML therapy.

Idarubicin Plus Azacitidine + Venetoclax As Induction Therapy Achieved High Remission in Elderly Fit- Acute Myeloid Leukemia Patients: Preliminary Results of a Phase II Study

Background The diagnosed age of de novo acute myeloid leukemia (AML) increased over the years, with a median current age of 68 years old. Elderly AML patients characterized by harboring high-risk mutations and significant comorbidities, usually display resistance to conventional chemotherapy, leading to poor outcomes. Although encouraging results from VIALE-A were reported in these years, over 30% participants had limited responses to the regimen of azacitidine + venetoclax after first-cycle induction. Thus, the selection of initial regimen for elderly AML is worth further exploration, especially for those with good fitness (the fitness was evaluated based on the physical ability, emotional status, cognitive function, and comorbidities). Here, we added idarubicin to azacitidine + venetoclax as the induction regimen (IAV regimen) for elderly fit-AML, and reported the preliminary results of efficacy and safety. Methods This single-center, single-arm, phase II trial (ChiCTR2300070190) was conducted at Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (Shanghai, China). Elderly patients (aged 60-75 years) of newly diagnosed acute myeloid leukemia (AML) were assessed via modified comprehensive geriatric assessment (mCGA) for the fitness. From April 2023, 22 patients have been enrolled for the treatment of IAV regimen (cycle 1: IDA 6 mg/m 2 d1-3, AZA 75 mg/m 2 d1-7, VEN 100 mg d1, 200 mg d2, 400 mg d3-14; cycle 2: IDA 6 mg/m 2 d1-2, AZA 75 mg/m 2 d1-5, VEN 400 mg d1-14). Among them, 19 patients and 13 patients who have completed cycle 1 and cycle 2 treatment, respectively, were available for analyzing the preliminary efficacy and safety in the trial. The primary objective of this study was to access the composite complete remission (cCR: CR + CRi) rate of IAV treatment. The secondary objective was to evaluate the MRD-negative rate, regimen-related toxicity, and safety. The duration of CR, overall survival rate and event-free survival rate would also be further accessed with sufficient follow-up time. Results The median age of currently enrolled patients was 64 (61-69) years old, and males accounted for 68.4%. Detailed baseline characters were summarized in Table 1. Composite complete remission rate after 1-cycle IAV treatment was achieved in 89.5% (17/19) participants, among which 52.9% (9/17) got MRD-negative status. One patient (1/19, 5.26%) got partial remission, with 7.5% blasts remained. After cycle 2 treatment, the CR rate increased to 92.9% (13/14), and 92.3% (12/13) CR participants were MRD-negative (Table 1). Till now, only 1 patient with chromothripsis-associated rearrangements showed no response and died on day 40 after treatment initiation. The median recovery time of neutrophils (ANC > 0.5×10 9/L) and platelet (PLT > 50×10 9/L) was 20.5 (16-30) days and 20.5 (15-43) days, respectively (Table 1). The most frequent grade 3/4 non-hematological adverse events (according to CTCAE version 5.0) were febrile neutropenia (grade 3, n = 14; grade 4, n = 5), pneumonia (grade 3, n = 5) and sepsis (grade 3, n = 2; grade 4, n = 1) (Table 2). Conclusion In this study, IAV regimen showed promising efficacy on elderly fit-AML patients, with high rates of MRD-negative remission. Non-hematological toxicities were tolerable under current observation. Based on the invigorating preliminary results, the efficacy and potential benefit of IAV regimen on elderly fit AML is worthy of follow-up in the future.

Folate-Appended Hydroxypropyl-β-Cyclodextrin Induces Autophagic Cell Death in Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is a heterogenous myeloid neoplasm that remains challenging to treat. Because intensive conventional chemotherapy reduces survival rates in elderly patients, drugs with lower toxicity and fewer side effects are needed urgently. 2-Hydroxypropyl-β-cyclodextrin (HP-β-CyD) is used clinically as a pharmaceutical excipient for poorly water-soluble drugs. Previously, we showed that HP-β-CyD exerts antitumor activity by disrupting cholesterol homeostasis. Recently, we developed folate-conjugated HP-β-CyD (FA-HP-β-CyD) and demonstrated its potential as a new antitumor agent that induces not only apoptosis, but also autophagic cell death; however, we do not know whether FA-HP-β-CyD exerts these effects against AML. Here, we investigated the effects of FA-HP-β-CyD on folate receptor (FR)-expressing AML cells. We found that the cytotoxic activity of FA-HP-β-CyD against AML cells was stronger than that of HP-β-CyD. Also, FA-HP-CyD induced the formation of autophagosomes in AML cell lines. FA-HP-β-CyD increased the inhibitory effects of cytarabine and a BCL-2-selective inhibitor, Venetoclax, which are commonly used treat elderly AML patients. Notably, FA-HP-β-CyD suppressed the proliferation of AML cells in BALB/c nude recombinase-activating gene-2 (Rag-2)/Janus kinase 3 (Jak3) double-deficient mice with AML. These results suggest that FA-HP-β-CyD acts as a potent anticancer agent for AML chemotherapy by regulating autophagy.

Bone marrow stromal cells reduce low-dose cytarabine-induced differentiation of acute myeloid leukemia.

Low-dose cytarabine (LDAC) is a standard therapy for elderly acute myeloid leukemia (AML) patients unfit for intensive chemotherapy. While high doses of cytarabine induce cytotoxicity, the precise mechanism of action of LDAC in AML remains elusive. In vitro studies have demonstrated LDAC-induced differentiation; however, such differentiation is seldom observed in vivo. We hypothesize that this discrepancy may be attributed to the influence of bone marrow (BM) stromal cells on AML cells. Thus, this study aimed to investigate the impact of BM stromal cells on LDAC-induced differentiation of AML cell lines and primary samples. Our results demonstrate that the presence of MS-5 stromal cells prevented LDAC-induced cell cycle arrest, DNA damage signaling and differentiation of U937 and MOLM-13 cell lines. Although transcriptomic analysis revealed that the stroma reduces the expression of genes involved in cytokine signaling and oxidative stress, data obtained with pharmacological inhibitors and neutralizing antibodies did not support the role for CXCL12, TGF-β1 or reactive oxygen species. The presence of stromal cells reduces LDAC-induced differentiation in primary samples from AML-M4 and myelodysplastic syndrome/AML patients. In conclusion, our study demonstrates that BM stroma reduces differentiation of AML induced by LDAC. These findings provide insights into the limited occurrence of terminal differentiation observed in AML patients, and suggest a potential explanation for this observation.

Clinical impact of the genomic landscape and leukemogenic trajectories in non-intensively treated elderly acute myeloid leukemia patients

To characterize the genomic landscape and leukemogenic pathways of older, newly diagnosed, non-intensively treated patients with AML and to study the clinical implications, comprehensive genetics analyses were performed including targeted DNA sequencing of 263 genes in 604 patients treated in a prospective Phase III clinical trial. Leukemic trajectories were delineated using oncogenetic tree modeling and hierarchical clustering, and prognostic groups were derived from multivariable Cox regression models. Clonal hematopoiesis-related genes (ASXL1, TET2, SRSF2, DNMT3A) were most frequently mutated. The oncogenetic modeling algorithm produced a tree with five branches with ASXL1, DDX41, DNMT3A, TET2, and TP53 emanating from the root suggesting leukemia-initiating events which gave rise to further subbranches with distinct subclones. Unsupervised clustering mirrored the genetic groups identified by the tree model. Multivariable analysis identified FLT3 internal tandem duplications (ITD), SRSF2, and TP53 mutations as poor prognostic factors, while DDX41 mutations exerted an exceptionally favorable effect. Subsequent backwards elimination based on the Akaike information criterion delineated three genetic risk groups: DDX41 mutations (favorable-risk), DDX41wildtype/FLT3-ITDneg/TP53wildtype (intermediate-risk), and FLT3-ITD or TP53 mutations (high-risk). Our data identified distinct trajectories of leukemia development in older AML patients and provide a basis for a clinically meaningful genetic outcome stratification for patients receiving less intensive therapies.

Analysis of single-cell sequencing results of an elderly patient with myeloid leukemia reveals high expression of multiple oncogenes in monocytes and hematopoietic stem cells

ABSTRACTObjectiveOlder patients with acute myeloid leukemia (AML) face a higher risk of death. This study analyzed the gene sequencing results of an elderly AML patient to provide new ideas for treatment.MethodsThis study performed single-cell RNA sequencing (scRNA-seq) bioinformatics analysis of blood cells in the peripheral blood and bone marrow of a 64-year-old AML-M5 patient before chemotherapy. The differentially expressed genes (DEGs) were identified, and functional enrichment analyses were performed.ResultsA total of 7990 and 123 DEGs were identified in monocytes and hematopoietic stem cells (HSCs), respectively. Among the top 40 DEGs analyzed, MYB showed high expression in peripheral blood monocytes, while 13 other tumor-related genes exhibited high expression in monocytes in the bone marrow. Peripheral blood and bone marrow HSCs had 6 and 12 highly expressed tumor-related genes respectively, including MCL1, JUN, and JUNB. These genes may form a interconnected network contributing to the progression and heterogeneity of AML, which can have an impact on patient treatment and prognosis.ConclusionsIn conclusion, when treating elderly AML patients, it is important to consider their individual characteristics in order to optimize treatment strategies.

Venous thromboembolism and acute myeloid leukemia: risk factors and mortality in elderly white, black and Asian patients.

Risk factors for venous thromboembolism (VTE) in elderly patients with acute myeloid leukemia (AML) are not known by race. The aim of this study was to determine the association of VTE with known risk factors and the impact of VTE on mortality in elderly white, black and Asian patients with AML. The merged SEER-Medicare database (2000-2015) was used for patients aged at least 65 years diagnosed with AML. Multivariable logistic regression was used to examine the association of VTE with known risk factors and Cox proportional hazards regression was used to evaluate the association of VTE with mortality in white, black and Asian patients. Among 21 403 AML patients aged at least 65years, VTE was diagnosed in 10.6% of 18 731 white patients, 13.4% of 1362 black and 5.6% of 1310 Asian patients. Overall, the adjusted risk of VTE in black patients was similar to white patients, but Asian patients had a lower risk of VTE. Risk factors for VTE in white patients were age less than 75 years, female sex, chemotherapy and comorbid medical conditions, including hypertension, anemia, chronic kidney and lung disease, hyperlipidemia, heart failure and obesity. In black patients, hyperlipidemia, and heart failure and in Asian patients, age less than 75 years, female sex, chemotherapy and hypertension and myocardial infarction were associated with VTE. Central venous catheter placement was a predictor of VTE in all three races. Our study identified risk factors for VTE by race in elderly white, black and Asian AML patients.